One of the applications of modern biotechnology has been to enable the genetic engineering of higher plants. By the term genetic engineering, as used here, it is intended to describe the insertion into the inheritable genetic material of a plant one or more foreign, usually chimeric, genes which are either not natively present in the plant genome or are not present in the plant in that form. The transformed plant itself and its progeny which carry the inserted gene are referred to as transgenic plants, and the inserted gene may sometimes be referred to as a transgene. It is important that the inserted foreign gene be inheritable by progeny of the original engineered plant by normal sexual Mendelian inheritance, in other words that the germ line of the plant be transformed.
The first, and still most widely used, method of the genetic engineering of plants is based on the ability of a natural plant pathogen, Agrobacterium tumefaciens, to insert a portion of its plasmid DNA, referred to at its T-DNA (transfer DNA), into a host plant. The Agrobacterium plasmid which is responsible for this ability is known as "Ti" plasmid, for tumor-inducing, since the native function of the plasmid is to induce in infected plant cells an oncogenic process which also produces metabolites on which the bacteria feeds. Scientists have learned how to remove the oncogenic capability from, or "disarm," the Ti plasmid from Agrobacterium, and then to insert into the T-DNA of the disarmed Ti plasmid the foreign gene which is sought to be inserted into the plant. The Agrobacterium carrying the altered Ti plasmid is then allowed to infect susceptible plant cells, and its transfer process carries the foreign gene or genes in the T-DNA into the plant cells. If a selectable resistance marker, i.e. a transgene which confers resistance to an antibiotic or herbicide to which the plant is susceptible, is incorporated into the Ti plasmid, the selection agent can be used to select for the transformed plant cells. The transformed cells can then be regenerated into whole sexually mature plants.
The techniques of Agrobacterium-mediated plant transformation have been applied to a large number of plants including tobacco, tomato, petunia, cotton, carrot, soybean and walnut. The technique may be limited in some plants, however, due to limitations in the host range of Agrobacterium species (notably to dicot plants) and the lack of regeneration protocols for some plants. Other approaches have enabled the genetic engineering of most of the important plant species not susceptible to Agrobacterium transformation. It is possible to introduce genes into individual plant cells by electroporation, involving electric shock, or by chemical cell wall disruption using polyethylene glycol, and these techniques have been used to transform protoplasts of rice and other cereals, which are not Agrobacterium hosts. For species which can be regenerated from protoplasts, this approach is practical. Another recently developed technique makes use of microprojectiles coated with DNA, which are physically accelerated into plant cells. This acceleration particle transformation technique has been reported to work with tissue cultures of tobacco, with suspension cultures of maize and cotton, and with meristematic tissue of soybean, poplar, and cotton.
In general, while transgenic plants are, of course, somewhat different from native plants of the species, they are generally not radically altered. The transgenic plants may carry one or more, sometimes many, copies of an inserted foreign gene. The inserted genes can often be expressed, although for the genes that are expressed, the level of expression will vary depending on variables such as copy number, site of insertion (which is believed random), strength of promoter or enhancers, and character of coding sequence. Since copy number and insertion site vary with each transformation event, it is usually the case that several independently transgenic plant families or lines are created, which may have slightly different expression characteristics. In general, there do not appear to be fundamental differences among the transgenic plants created by any of these methods, i.e. there is variation in the plants, but it is independent of the method of transformation. In any event, while not all plants have yet been genetically engineered, the presently available techniques, and the wide variety of plants to which they have been applied, suggest that there are no biological barriers to the genetic engineering of any plant species.
In the study of transgenic plants, tobacco and Arapidopsis are often used as model systems. This is because tobacco is generally one of the easiest plants to genetically engineer by Agrobacterium transformation, due to the availability well-known and convenient selectable markers, and ready regeneration protocols. In general, transgenes which have been expressed well in tobacco have been demonstrated to express with similar characteristics in other plant species. Tobacco is also typical of stress sensitive crop plants in its osmotic regulation and sugar synthesis. Tobacco does not natively produce mannitol in its tissues and has been reported to be unable to metabolize mannitol.
While the procedures for the genetic engineering of most of the important agricultural crop species have now been developed, there has been somewhat less progress in the identification of what foreign traits or genes may be usefully inserted into plants. The best known examples so far in the technology involve genes which confer resistances, for example resistances to herbicides or to pests. Such genes can confer the desired trait (i.e. resistance) with a single transgene. To improve some of the more agronomically important traits of plants relating to vigor, yield, water or salt tolerance, heat stress, or the like, appears initially to be a more difficult objective. The traits which are associated with these qualities are poorly understood, and the gene, or more likely, genes, associated with the various traits are generally uncharacterized. Accordingly, there is a need to identify new classes of traits, or genes, which can be inserted into crop plants to attempt to make them grow better. Even if newly inserted genes do not make a plant perform better in agricultural conditions, transgenic plants carrying such genes are useful for research purposes for investigating how changes in plant internal processes (e.g. osmotic regulation) affect the field performance of the plants.
All plants, of course, capture energy in the form of sunlight and store energy in a chemical form as sugars. However, the sugars which plants manufacture vary in kind and relative amounts from plant to plant. In addition to serving their function of chemical energy storage, some sugars or other carbohydrates may also serve to regulate the osmotic balance of the plants. The osmotic ability of the plant cells, and the relative osmotic balances among the subcellular organelles, may be fundamentally related to the ability of plants to withstand stresses of a variety of types, such as freezing or salt stress, in addition to drought or water stress. Cold, for example, may be fatal to plant tissues due to water loss long before temperature extremes are reached at which ice would crystallize inside plant tissues. This ability to withstand water stress may be fundamentally related to plant performance in adverse conditions.
The role of polyalcohol sugars, or polyols, in plant metabolism is poorly understood, in spite of the fact that up to 30% of the annual global carbon production by higher plants may go into polyols rather than simple sugars. Of the polyols, mannitol is the most abundant in nature. While it is found in about seventy plant families, it is not produced at detectable levels in any important agricultural field or vegetable crop, other than celery (Apiaceae), coffee (Rubiaceae) and olive (Oleacea). Mannitol is quite commonly produced in algae and fungi.
Other polyols are common in some plant species, even in some instances in which no metabolic role for polyols are apparent. For example, the polyols ononitol and pinitol are known to be produced in some plants under conditions of stress from drought, salt or low temperature. In some of these plants, the polyol produced appears to be a dead-end product, i.e. one which has no further metabolic role and from which no other metabolite is synthesized. This raises the possibility that the accumulation of such polyols serves an osmotic regulatory role.
In plants, there are two separate pathways for mannitol biosynthesis. One pathway used, for example in brown algae, proceeds from the reduction of fructose-6-P to mannitol-1-P by mannitol-1-P dehydrogenase, with an NAD cofactor, followed by dephosphorylation by a specific mannitol-1-P phosphatase. (Mannitol-6-P and mannitol-1-P are synonymous.) In celery, the process is different, beginning with mannose-6-P, which is reduced to mannitol-1-P by mannose-6-P reductase with an NADP cofactor, followed again by dephosphorylation.
In E. coli, a mannitol catabolic system is known. In E. coli, mannitol is taken in from the environment and converted by phosphorylation to mannitol-1-phosphate (M1P). Then the NAD dependent enzyme, mannitol 1-phosphate dehydrogenase, (M1PD) converts the mannitol-1-phosphate to fructose 6-phosphate in an equilibrium reaction. The gene coding for this enzyme, referred to as mt1D, has been previously cloned by others.
One approach to evaluate the role of polyols in plant stress response is to examine polyol production in stress tolerant plants. There are a number of salt tolerant plants, referred to as halophytes, which are relatively tolerant to drought and cold, as well as salt. Unfortunately, most of our important crop plants are salt-sensitive species, referred to as glycophytes. If the genes and mechanisms used by halophytes to combat stress are identified, it may become possible to transfer those genes and/or mechanisms into important crop plants by genetic engineering.
One unique system that can be used to identify stress tolerance genes and mechanisms is the inducible halophyte, Mesembryanthemum crystallinum, the common ice plant. As a facultative halophyte, the ice plant undergoes a set of stress-induced biochemical changes to become more stress tolerant. One of those changes involve a switch of metabolic pathways, i.e. from C.sub.3 to crassulacean acid metabolism, as a water conservation measure. Others of those changes were heretofore poorly characterized.